DNA methylation is an important regulator of gene expression and may play a role in the development and progression of a number of diseases, such as cancer. Methylation is typically limited to cytosines located 5′ to a guanine (i.e., CpG sequences), however other forms of methylation are known. Research suggests that genes with high levels of methylation in a promoter region are transcriptionally silent, which may allow unchecked cell proliferation.
When a promoter region has excessive methylation, the methylation is typically most prevalent in sequences having CpG repeats, so called “CpG islands.” Undermethylation (hypomethylation) has also been implicated in the development and progression of cancer through different mechanisms.
Several methods have been developed to identify and quantify methylation, especially in CpG sites, e.g., CpG islands, that are implicated in silencing promoters. Those include sequencing methods in which genomic DNA is isolated and treated with bisulfite. Because methylated cytosines are not affected by bisulfite treatment, the unmethylated Cs, e.g., within a CpG site, are converted to uracil, while methylated Cs are not converted. After sequencing, comparison of the starting DNA and the bisulfate treated DNA indicates the location of methylation sites.
Perhaps the most widely-used method of probing methylation patterns is methylation specific PCR (MSP) which uses two sets of primers for an amplification reaction. One primer set is complimentary to sequences whose Cs are converted to Us by bisulfite treatment, and the other primer set is complimentary to non-converted Cs. Using these two separate primer sets, both the methylated and unmethylated DNA are amplified. Comparison of the amplification products gives insight as to the methylation in a given sequence. See Herman et al., “Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands,” P.N.A.S., vol. 93, p. 9821-26 (1996), which is incorporated herein by reference in its entirety. This technique can detect methylation changes as small as ±0.1%. In addition to methylation of CpG islands, many of the sequences surrounding clinically relevant hypermethylated CpG islands can also be hypermethylated, and are potential biomarkers.
A problem with MSP is that it requires the use of two different primer sets, one for sequences containing methylated Cs, and the other for sequences containing Us, which were converted from unmethylated Cs by bisulfite treatment. Using two different primer sets limits the application of MSP. In addition to the costs associated with producing and maintaining two separate primer sets, the amplification process cannot be maximally efficient because of the need to operate in temperature regimes appropriate for both primer sets.